ABSTRACT One of the biggest breakthroughs in cancer biology in the last decade has been the development of small-molecule kinase inhibitors, with more than >150 kinase inhibitors in clinical use or clinical development that target one of the >500 kinases found in human cells. Despite these successes, the development of kinase inhibitors with high specificity remains a challenge; combined with the problem of resistance, this argues that identification of a different class of kinases inhibitors is needed. This application is directed at an alternative mode of kinase inhibition, by testing the possibility that there are as-yet-undiscovered protein surfaces that could provide alternative targets for in vivo inhibition of kinase function. To pursue this premise, a novel methodology that provides a robust and rapid means of identifying novel mutations in functionally important amino acids on protein surfaces will be pursued. The methodology, which employs large-scale mutagenesis screens in yeast, with be applied to a large collection of yeast protein kinases, with the goal of identifying novel regulatory activities for each kinase. The kinases to be screened will be those for which there is prior data supporting an oncogenic role in human cells, and sufficient sequence similarity between the human and yeast orthologs to allow comparable missense mutations to be generated in the human protein. Mutations recovered from this screen will be subsequently analyzed in yeast in order to identify pathway-specific, and potentially novel, activities, as a precursor to whether the comparable mutations, when introduced into human kinases, can inhibit signaling and/or proliferation in cancer cell lines. The overall goal will be to identify new candidates as the basis for screens for small molecule therapeutics, that will provide treatment options that confer increased selectivity and specificity, by combining inhibitors that target both enzyme activity and other specificity determinants.